Methods for the preparation, isolation and purification of epothilone B, and x-ray crystal structures of epothilone B

ABSTRACT

The present invention relates to improved methods for the production, isolation and purification of epothilone B. These methods include, for example, a fermentation process for the production of epothilone B, isolation via adsorption onto a resin, and subsequent purification.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/412,994 filed Sep. 23, 2002.

FIELD OF INVENTION

The present invention relates to improved methods for the production,isolation and purification of epothilone B. These methods include, forexample, a fermentation process for the production of epothilone B,isolation via adsorption onto a resin, and subsequent purification.

BACKGROUND OF INVENTION

Epothilones are a relatively new class of macrolide compounds that wereoriginally obtained by fermentation of myxobacteria (Sorangiumcellulosum). These compounds were initially investigated as plantprotective agents due to their anti-fungal properties. Epothilones thenbecame of interest due to their cytotoxic activity on animal cells, andwere subsequently characterized as tubulin polymerization agents. It isnow known that epothilones exert microtubule-stabilizing effects similarto paclitaxel (TAXOL®) and cytotoxic activity against rapidlyproliferating cells such as tumor cells or other hyperproliferativecellular disease. The use of epothilones as chemotherapeutic agents isdescribed in Bollag et al., Cancer Research 55, 2325, 1995.

Epothilones A and B (epo A or epo B, respectively) have the structures,

-   -   Epothilone A R═H    -   Epothilone B R═Me

One scheme for obtaining epothilones was revealed by Höfle et al. in WO93/10121. Höfle cultured a strain of Sorangium cellulosum in a mediumcontaining carbon sources, nitrogen sources and mineral salts. Anadsorber resin was added during the culturing of the strain. Theepothilones were eluted with solvent from the adsorbent resin. Thevarious epothilones were separated by reverse-phase chromatography andcrystallized. However, Höfle et al. conceded that this method producedonly a low quantity of epothilone B, and also that the ratio ofepothilone B to epothilone A in the fermentation was low. This low ratioof epothilone B relative to epothilone A makes recovery of pureepothilone B difficult. Thus, there is a need in the art for improvedmethods of fermentation to produce epothilone B in preference toepothilone A, and improved methods of isolation and purification ofepothilone B.

SUMMARY OF INVENTION

The present invention is directed to an improved fermentation processfor the production of epothilone B.

Further included in the present invention are new strains of Sorangiumcellulosum obtained by mutagenesis for the production of epothilones.

Also included in the present invention are methods to improve the ratioof epothilone B to A produced by the new strain of Sorangium cellulosumby providing an additive to the fermentation. In one preferredembodiment, the additive is propionate, propionic acid with proper pHadjustment, or another propionate precursor.

Also included in the present invention is an improved extraction processfor isolation of epothilone B from the fermentation medium using aresin. Further included are methods for washing epothilone-rich resin toreduce impurity levels and improve downstream processing.

Also included in the present invention is an improved process for thepurification of epothilone B. In one embodiment, purification isachieved using crystallization. In another embodiment, purification isachieved by chromatographic methods which include normal-phasechromatography or reverse-phase chromatography. In yet anotherembodiment, purification is achieved by a combination of crystallizationand purification of samples by chromatography, including normal andreverse-phase chromatography. In a further embodiment, the resin extractis processed by crystallization only.

Epothilone B (“epo B”) is useful as an intermediate in the preparationof derivative 1 (“D1”), (as described in U.S. Pat. No. 6,262,094, hereinincorporated by reference), where the 2-methyl on the thiazole ring issubstituted with an amine:

Epothilone B is also useful in the preparation of derivative 2 (“D2”)(such conversion of the lactone of epothilone B to the lactam ofderivative 2 is described by Borzilleri et al., J. Amer. Chem. Soc. 122,8890, 2000, and in WO 99/02514, herein incorporated by reference):

Furthermore, epothilone B (“epo B”) is useful for the preparation ofderivative 3 (epothilone D, “D3”) (as described in U.S. Pat. No.6,320,045, herein incorporated by reference):

Further included in the invention are crystal forms of epothilone Bproduced using the methods and materials described herein.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary, but not restrictive,of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The advantages, nature and various features of the invention may appearmore fully upon consideration of the accompanying drawings. In thedrawings:

FIG. 1 shows the molecular structure in the monoclinic unit cell of formepoB-EAβ, with two molecules of epothilone B and two molecules of ethylacetate in the guest channel of the monoclinic unit cell.

FIG. 2 shows the molecular structure in the monoclinic unit cell of formepoB-ANβ, with two molecules of epothilone B and two molecules ofacetonitrile in the guest channel of the monoclinic unit cell.

FIG. 3 shows the molecular structure in the monoclinic unit cell of formepoB-Ipβ, with two molecules of epothilone B and two molecules ofisopropanol in the guest channel of the monoclinic unit cell.

FIG. 4 shows the molecular structure in the monoclinic unit cell of formepoB-Toβ, with two molecules of epothilone B and two molecules oftoluene in the guest channel of the monoclinic unit cell.

FIG. 5 shows observed (top) and simulated (bottom) PXRD patterns for theethyl acetate solvate (crystal form epoB-EAβ) of epothilone B. In FIG.5, the simulated pattern was calculated from the refined atomicparameters in the monoclinic crystal structure at −33° C., and theobserved pattern was measured at +23° C.

FIG. 6 shows observed (top) and simulated (bottom) PXRD patterns for thetoluene solvate (crystal form epoB-TOβ) of epothilone B. In FIG. 6, thesimulated pattern was calculated from the refined atomic parameters inthe monoclinic crystal structure at −33° C., and the observed patternwas measured at +23° C.

FIG. 7 shows observed (top) and simulated (bottom) PXRD patterns for theacetonitrile solvate (crystal form epoB-ANβ) of epothilone B. In FIG. 7,the simulated pattern was calculated from the refined atomic parametersin the monoclinic crystal structure at −40° C., and the observed patternwas measured at +23° C.

FIG. 8 shows observed (top) and simulated (bottom) PXRD patterns for theisopropyl alcohol solvate (crystal form epoB-IPβ) of epothilone B. InFIG. 8, the simulated pattern was calculated from the refined atomicparameters in the monoclinic crystal structure at −30° C., and theobserved pattern was measured at +23° C.

FIG. 9 shows an observed PXRD pattern for a toluene-containing primarygrade solvate of epothilone B produced following the method described inExample 7, Step A.

FIG. 10 shows the thermal analysis (DSC and TGA) for thetoluene-containing primary grade solvate of FIG. 9.

FIG. 11 shows an observed PXRD pattern for a toluene-containingrecrystallized solvate of epothilone B, produced following the methoddescribed in Example 7, Step B.

FIG. 12 shows the thermal analysis (DSC and TGA) for thetoluene-containing recrystallized solvate of FIG. 11.

FIG. 13 shows an observed PXRD pattern for the ethyl acetate containingsolvate of epothilone B, produced following the method described inExample 7, Step C.

FIG. 14 shows the thermal analysis (DSC and TGA) for the ethyl acetatecontaining solvate of FIG. 13.

FIG. 15 shows an observed (top) PXRD pattern for the toluene-containingsolvate prepared following the method described in Example 7C, togetherwith a simulated (bottom) PXRD pattern for the toluene solvate ofepothilone B at room temperature.

FIG. 16 shows the thermal analysis (DSC and TGA) for thetoluene-containing solvate of FIG. 15.

It is to be understood that these drawings are for purposes ofillustrating the concepts of the invention and are not limiting innature. In each of FIGS. 1 through 4, all methyl and methylene hydrogenatoms of the epothilone have been omitted for clarity. In FIGS. 1–4,intermolecular hydrogen bonds are shown at the bottom right and top leftportions of the diagrams as dashed rods, and H-bond distances(Angstroms) designate the intermolecular oxygen—oxygen distances.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes specific process methods and novelmutant strains of Sorangium cellulosum, which together or separatelyproduce fermentations with improved concentrations of epothilone B,primarily by reduction of the relative amount of epothilone A producedduring the fermentation. Cells of Sorangium cellulosum or otherappropriate microorganisms are, for example, expanded through one ormore initial growth stage cultivations, and used to provide inoculum forepothilone-producing fermentations. During the first hours offermentation, for example in the neighborhood of 24–72 hours, cellgrowth occurs as the cells utilize nutrients in the medium. Thereafter,nutrients, such as vitamins, minerals, carbohydrates and amino acids (orother carbon or nitrogen sources such as amino acid precursors), areadded to the medium in an amount conducive to production of epothilones.In one embodiment, the nutrients, such as vitamins, minerals,carbohydrates and amino acids are added in an amount which maintains themaximum production rate of epothilone A or epothilone B during thefermentation. In one embodiment, the maximum production rate ofepothilone A or epothilone B is a production rate in which a greateramount of epothilone A or epothilone B is produced as compared to thatproduced without the addition of additives or nutrients or also resultsin a greater production rate that would occur if the additives ornutrients were added in an amount that is less than optimal. During thefermentation, propionic acid, a precursor thereof, or a salt thereof, isadded in an amount effective to increase the epothilone B to epothiloneA ratio (the “product ratio”).

The present invention is also directed to new strains of Sorangiumcellulosum which are useful in the manufacture of the epothilones. Thesenew strains, particularly strain SC16408, have been obtained bymutagenesis followed by random selection.

Sorangium cellulosum was first isolated from a soil sample collectedfrom the banks of the Zambesi River in South Africa in 1985. Theorganism was first described for production of the epothilones by Höfleet al. (cited above). The strain used by Höfle, et al. was designated Soce90, and is deposited at the Deutsche Sammlung von Mikroorganismen(German Collection of Microorganisms, DSM) under Deposit No. 6773.Strain So ce90 was subjected to UV mutagenesis followed by randomselection to generate strain So ce90B2. Strain So ce90B2 (alsodesignated SC16224) yielded epothilone B titers in shake flasks(containing for example 1.8 w/v % resin per flask) of approximately 50mg/L or 2.8 mg/g resin (which can for example range to 3.5 or 4.5 mg/g),and a ratio of epothilone B/A of approximately 0.6.

In the present invention, strain So ce90B2 or a derivative thereof wassubjected to mutagenesis with nitrosoguanidine (NTG), followed by randomselection to produce strains SC16408 (which is deposited as ATCC No.PTA-3880) and SC16449 (which is deposited as ATCC No. PTA-3881). Theselatter two strains have been deposited with the American Type CultureCollection as patent deposits pursuant to the Budapest Treaty. Detailsof the selection process are set forth in the examples.

The present invention provides, in one embodiment, Sorangium cellulosumstrains that produce (e.g., under production conditions defined below)at least about 100 mg of epothilone B per liter of broth volume. Inanother embodiment, the invention provides strains that produce, underepothilone B comparative production conditions, at least 80 mg ofepothilone B per liter of broth volume and an epothilone B to epothiloneA ratio of at least 1. The present invention provides in one embodimentstrains that produce 5 mg of epothilone B/g of resin of epothilone B, or5 mg/g resin at an epothilone B/A ratio of at least 1.0. In anotherembodiment, the epothilone B/A ratio is at least 1.5. In yet anotherembodiment, the epothilone B/A ratio is 1.5 to 4.0.

The present invention is also directed to methods to improve the ratioof epothilone B to A produced by Sorangium cellulosum by feeding anadditive to the fermentation. In preferred embodiments, the additivecomprises propionate, added after cells have grown for up to 96 hours,but preferably at approximately 24–48 hours. In some preferredembodiments, the cells were grown for approximately 34 hours beforepropionate was added. Early studies by GBF investigated, among otherfactors influencing fermentation, the effect of a one-time propionateaddition to the medium at a level of 0.1% for incremental improvement inthe epothilone B/epothilone A (B/A) ratio. Inventors herein have found,surprisingly, and it is one of the features of the present invention,that the titers of the epothilones, epothilone B in particular, and theB/A ratio produced in shake flasks, 14 L fermentors and productionfermentors, were improved markedly by the feeding of propionate orsodium propionate. Feeding of propionate or sodium propionate producessignificant improvement of epothilone B titers. For example, flaskproduction of epothilone B was improved by supplementation with sodiumpropionate to within a preferred range, as monitored in the culture, of0.05 to 0.80 mg/mL (0.005–0.08%) periodically (e.g., per day) oncefeeding was initiated, more preferably within a range of 0.005–0.04%. Inone embodiment, the amount of propionate in the culture is targeted to0.02% or less. In addition, other propionate-related compoundsincluding, but not limited to propionic acid methyl ester and propionicacid ethyl ester, were also found to improve epothilone B production andsubsequently the B/A ratio.

In one embodiment, particularly useful for fermentations in a flask, anadditional feed containing a mixture of monobasic and dibasic phosphateis added, with the ratio selected to support an appropriate pH. Thisfeed can be incorporated into a propionate feed or added separately.

In the present invention, a method of large scale epothilonepurification is described which successfully utilizes resin addition.The inclusion of resin was found to be useful in the isolation andpurification of epothilones, and also to dramatically improve theepothilone titers. In one preferred embodiment of the present invention,the resin is a styrene/divinylbenzene polymeric resin, such as an XADresin, preferably XAD-16 or the equivalent (available as AmberliteXAD-16 from Sigma-Aldrich, St. Louis, Mo. or Rohm and Haas Co.,Philadelphia, Pa.). Other Amberlite resins with hydrophobic surfaces,such as styrene-based XAD-4, XAD-1180 or XAD-1600 (Rohm and Haas Co.)can also be useful in the invention, as well as resins such asstyrene-based XD-207, HP20, HP21, SP825, SP850, SP700 or SP207 (which ismore hydrophobic due to added bromine groups) (these resins are fromMitsubishi, Tokyo, Japan or Mitsubishi Chemical America, Inc., WhitePlains, N.Y.). The resin can be incorporated into the medium within abroad range, such as 0.2 w/v % to 5.0 w/v %, and preferably 1.5 w/v % to4.0 w/v %.

The resin containing epothilones from the fermentation is optionallywashed with water and either 20–30% aqueous acetonitrile or aqueousmethanol to remove polar impurities, or with a solution containingdetergent, preferably an ionic detergent such as an alkyl sulfate-baseddetergent, and an amount of an amine (added to the solution in baseform). Amounts are selected to improve the quality of the epothiloneextract obtained later from the resin. One preferred aqueous wash uses0.5 w/v % sodium dodecyl sulfate and 0.5 w/v % ammonia. In this lastembodiment, prior to solvent extraction the resin is preferably washedone or more times with water.

The resin containing epothilones from the fermentation is preferablyextracted with a solvent that is immiscible with (phase-separates from)a water phase, such as ethyl acetate or methyl-t-butyl ether (MTBE), toremove epothilones adsorbed on the resin. Additional solvents that maybe useful for extracting epothilone B include n-butanol, isopropylacetate, n-propyl acetate, n-butyl acetate and t-butyl acetate. The richsolvent extract is preferably concentrated, and epothilone B iscrystallized from the concentrate. In one embodiment, the rich solventis washed with water, and the water-washed rich solvent is concentratedand optionally polish filtered. When the solvent is suitable, as isethyl acetate, epothilone B is crystallized by performing a distillativesolvent swap into an anti-solvent. In other words, a relativelyhigh-boiling-point second solvent in which epothilone B is essentiallyinsoluble is added to the rich solvent, and the rich solvent isdistilled away to a sufficient degree to allow crystallization. Vacuumcan be used to drive or facilitate the distillation. In one embodiment,the solvent is concentrated and a suitable amount of anti-solvent isadded. Useful anti-solvents include toluene, hexanes and heptanes. Theresulting slurry can be heated, and cooled to a set temperature selectedto enhance the quality of the resulting crystals. Temperatureoscillations can be used to improve crystal purity, minimize fines, andproduce a faster-filtering slurry. For some other solvents, such asMTBE, distillative concentration of the rich solvent produces aneffective crystallization environment on cooling (without the use of ananti-solvent). The resulting crystals are preferably filtered to yield aprimary grade epothilone B.

During the extraction and initial crystallization epothilone B isseparated from most impurities present in the initial extract,especially epothilone A. Primary grade epothilone B typically containsepothilone A as a major impurity. Also typically present are two otherstructurally similar impurities derived from the fermentation, namelythe following oxazole analogue and the ethyl thiazole analogue:

Subsequently applied purification methods (including recrystallizationand chromatography steps) described herein for epothilone B involve,among other things, the removal of these two compounds to a level wherethey are no longer considered significant.

Primary grade epothilone B (i.e., toluene-containing crystal form epo B,primary grade), preferably obtained as described above, can then berecrystallized by heating in ethyl acetate followed by the addition oftoluene with continued heating. The mixture is then cooled, theresulting crystalline slurry is filtered and the cake washed withtoluene to give once recrystallized epothilone B (i.e.,toluene-containing crystal form epo B, recrystallized) Alternatively,primary grade epothilone B can be processed through a preparative highperformance reverse-phase chromatography step (e.g., on RP/C-18 in theform of a column) as set forth in the examples. Optionally, prior toloading the epothilone sample onto the column, a preceding volume of asuitable organic solvent, or a mixture of organic solvents, is added toreduce precipitation of the epothilone. In one embodiment, the organicsolvent is one such as dimethylsulfoxide (DMSO). Optionally, a trailingvolume of a suitable organic solvent or a mixture of organic solvents isadded to reduce precipitation of the epothilone. Epothilones are theneluted with a suitable organic solvent, a mixture of organic solvents oran aqueous solution of an organic solvent. In one embodiment, theepothilones are eluted with a mixture of acetonitrile and water. Theelution profile using these solvents can, for example, be linear orgradient, and is chosen to obtain low impurity levels. Fractionscontaining epothilone B of desired purity are pooled, concentrated, andextracted with a solvent including, but not limited to, ethyl acetate.The rich solvent extracts are then concentrated and crystallized, forexample, by the addition of a low-polarity solvent such as n-heptane orheptanes, and optionally cooled. The slurry is filtered, washed withsolvent/anti-solvent (in a ratio and amount selected to not dissolvesignificant amounts of epothilone B), such as ethyl acetate/n-heptane ina 2:1 ratio. The washed crystals are dried to yield high-qualityepothilone B.

Other purification methods can be used, such as chromatography on normalphases such as silica, or silica based normal phases and the like. Forexample, high performance normal phase chromatography can be used.Samples can be loaded onto the column in a relatively low-polaritysolvent such as methylene chloride, and the epothilones eluted withhigher-polarity solvent, such as a mixture of ethyl acetate and heptane.The elution profile using these solvents can, for example, be linear orgradient, and is chosen to obtain low impurity levels. The desiredfractions are pooled, concentrated and crystallized, for example fromethyl acetate by the addition of a low-polarity solvent such asn-heptane, heptanes, or toluene. The slurry is filtered, washed withsolvent /anti-solvent (in a ratio and amount selected to not dissolvesignificant amounts of epothilone B), such as ethyl acetate/n-heptane ina 2:1 ratio or ethyl acetate/toluene. The washed crystals are dried toyield high quality epothilone B.

In certain cases where extensive removal of the ethyl thiazole oroxazole analogs is not required, such as in the synthesis of D1,epothilone B can be purified by crystallization alone. Solid epothiloneB material is dissolved, for example, in warm ethyl acetate andcrystallized (or recrystallized) by cooling to ambient temperature orcooler, followed by filtration and drying (e.g., in vacua).Crystallizations can be repeated to obtain the desired purity, such as 2to 3 times.

Growth medium for growing the epothilone-producing microorganism can be,for example, formulated as follows:

More Still More Preferred Preferred Preferred Ingredient (g/L) (g/L)(g/L) Powdered 0.5–12 1–8 2–6 Skim Milk Toasted 0.5–12 1–8 2–6 NutrisoyFlour¹ Tastone-154¹ 0.5–12 1–6 1–4 Maltrin-M040¹   4–18  6–14  8–12CaCl₂.2H₂O  0.2–2.4 0.4–1.6 0.8–1.2 MgSO₄.7H₂O  0.2–2.4 0.4–1.6 0.8–1.2EDTA, FeIII,  0.002–0.02  0.004–0.016 0.006–0.014 Na salt HEPES   6–20 8–16 10–14 Glycerol 0.5–12 1–8 2–6 ¹Other skim milk, soy flours, yeastextracts and Maltrin starches have also been used interchangeable withcomparable results.

Production medium for growing the epothilone-producing microorganism andfor production of epothilones, especially in shake flasks, can be forexample formulated as above with the following difference with respectto glycerol, and the following addition of resin:

More Still More Preferred Preferred Preferred Ingredient (g/L) (g/L)(g/L) Glycerol  2–20  4–16  6–14 Resin 10–40 12–35 15–30

A useful nutrient feed solution, especially for use in shake flasks,comprises:

Preferred Ingredient (%) Sodium 2–5 Propionate Maltrin-M040  8–12Tastone-154 2–5

Such nutrient feed can further contain a mixture of dibasic sodiumphosphate and monosodium phosphate, as follows:

Preferred Ingredient (%) Disodium phosphate 1.0–2.0 Monosodium phosphate0.3–0.7The ratio of disodium phosphate to monosodium phosphate is selected tominimize pH drift of the culture away from the desired pH upon additionof feed.

For use in fermentors, the nutrient components described above, with theexception of HEPES, which is preferably deleted, can preferably be usedwith antifoam (e.g., from Dow Corning, AF Emulsion, Food Grade) added asfollows:

More Still More Preferred Preferred Preferred Ingredient (g/L) (g/L)(g/L) Antifoam 0.5–5 1–4.5 1.5–4

Caustic (sodium or potassium hydroxide solution) can be added to thefermentation medium as needed to maintain a useful pH range. Resin canbe added as follows:

More Still More Preferred Preferred Preferred Ingredient (g/L) (g/L)(g/L) Resin 10–50 12–45 15–40

In the production fermentation, propionate and nutrients are preferablyadded separately as needed. Propionate feed can, for example, comprise80 to 150 g/L sodium propionate, with the amount most preferably addedto maintain (e.g., as determined by HPLC) propionate levels of 0.05 to0.20 mg/mL. Propionate addition can be initiated 20–40 hours afteradding the seed culture into the fermentor. The nutrients aresupplemented, for example, with a sterile feeder stock as follows:

Preferred Ingredient (g/L) Tastone-154 15–25 Maltrin-M040 55–75 Antifoam0.5–1.5

For longer term fermentations, additional nutrients are preferablyadded, for example, from the following sterile feeder stock, which isadded in higher volume compared to the preceding feeder stock:

Preferred Ingredient (g/L) Powdered Skim 40–60 Milk Maltrin-M040 140–180Glycerol 60–90 Antifoam 0.5–1.5

These nutrients of the above two feeds can be selected to avoidinitiating a growth phase.

The present invention includes processes for the production ofepothilone B wherein the epothilone B (“epo B”) is converted toDerivative 1 (“D1”) as described in U.S. Pat. No. 6,262,094, hereinincorporated by reference), having the following formula:

The present invention also includes processes for the production ofepothilone B wherein the epothilone B is converted to Derivative 2(“D2”) (described by Borzilleri et al., J. Amer. Chem. Soc. 122, 8890,2000, and in WO 99/02514, herein incorporated by reference), having theformula:

The present invention further includes processes for the production ofepothilone B wherein the epothilone B (“epo B”) is converted toDerivative 3 (epothilone D, “D3”) (as described in U.S. Pat. No.6,320,045, herein incorporated by reference), having the followingformula:

Crystal Forms of Epothilone B

Applicants also have made various crystal forms of epothilone B usingthe inventive methods and materials described herein. Epothilone Bcrystals have been obtained using different solvents and solventsystems. For example, applicants have discovered a toluene-containingepothilone B solvated crystal form, designated herein as epoB-Toβ,having the unit cell data reported below in Table 1. Thetoluene-containing solvated crystal form of epothilone B is furtherillustrated with FIGS. 9 through 12 and FIGS. 15 and 16 herein.Applicants also have obtained epothilone B crystals using acetonitrile(i.e., epoB-ANβ), ethyl acetate (i.e., epoB-Eaβ), and isopropyl alcohol(i.e., epoB-Ipβ), as well as the solvent systems described below in theexamples. These crystallographically isostructural forms have amonoclinic clathrate structure with a P2₁ space group containinglipophilic solvent channels that extend along the b-axis throughout thecrystals (1 channel/unit cell). Each channel can contain up to twosolvent molecules such as toluene, acetonitrile, ethyl acetate,isopropyl alcohol, or MTBE (ideally resulting in 1:1 solvates ofepothilone B). Crystallization from toluene/ethyl acetate solventmixtures (e.g., 1:1 mixture) results in preferential incorporation oftoluene in the clathrate channels (i.e., obtain form epoB-TOβ, notepoB-EAβ). Both hydrogen-bond donors of the epothilone (hydroxyls) areinvolved in interepothilone hydrogen bonds and are not available to bindto, and constrain, the guest solvents.

Forms epoB-TOβ, epoB-ANβ, epoB-EAβ, and epoB-IPβ display the unit celldata presented in Table 1. Crystallization conditions for obtainingthese forms of crystals containing toluene, acetonitrile, ethyl acetateand isopropanol are presented below in the examples. PXRD patterns forthe crystals prepared using the methods described in Example 7 are setforth in FIGS. 9, 11, and 13, also as further described below.

Tabulated specific exemplary parameters for these crystal forms are asfollows, and as shown in Table 1:

Form epoB-Toβ Crystallized from toluene as described in Example 8A.epoB-ANβ Crystallized from acetonitrile as described in Example 8B.epoB-EAβ Crystallized from ethyl acetate (EtOAc) as described in Example8C. epoB-IPβ Crystallized from isopropyl alcohol (IPA) as described inExample 8D.

Fractional atomic coordinates for epoB-ANβ, epoB-EAβ, epoB-IPβ andepoB-Toβ are shown in Tables 2, 3, 4 and 5, respectively. The PXRDpatterns set forth in FIGS. 9, 11 and 13 are characterized by the datalisted in Tables 6, 7 and 8, below.

TABLE 1 Unit cell data Form T(° C.) a(Å) b(Å) c(Å) B V Z V/Z sg d(calc)₁R Ideal solvent sites Epo B-TOβ −33 11.853(1) 10.613(2) 14.328(2)113.04(1) 1659(1) 2 829 P2₁ 1.201 0.09 1 toluene per epo B Epo B-ANβ −4011.961(1) 10.543(1) 13.601(2) 111.89(1) 1592(1) 2 796 P2₁ 1.145 0.07 1acetonitile per epo B Epo B-EAβ −33 11.939(1) 10.587(1) 13.882(1)111.87(1) 1628(1) 2 814 P2₁ 1.215 0.07 1 EtOAc per epo B EpoB-Ipβ −311.928(2) 10.610(1) 13.870(2) 111.92(1) 1628(1) 2 814 P2₁ 1.158 0.09 1IPA per epo B ¹Ideal densities calculated. assuming 1:1 solventoccupancy.

TABLE 2 Fractional Atomic Coordinates for Epothilone B AcetonitrileSolvate, Form EpoB-ANβ (most hydrogen atoms have been omitted) Atom X YZ C1 0.4422 0.2380 0.3076 C2 0.5619 0.2882 0.3165 C3 0.6422 0.18330.3013 C4 0.7565 0.2263 0.2807 C5 0.8531 0.2847 0.3800 C6 0.8409 0.41800.4193 C7 0.8968 0.4218 0.5419 C8 0.8360 0.3345 0.5975 C9 0.7205 0.39350.6018 C10 0.6345 0.2947 0.6184 C11 0.5287 0.3609 0.6355 C12 0.43280.2722 0.6397 C13 0.3118 0.2674 0.5573 C14 0.2626 0.3435 0.4562 C150.2748 0.2789 0.3613 O16 0.3977 0.3084 0.3684 C16 0.7197 0.3194 0.1878C17 0.8080 0.1051 0.2508 C18 0.9083 0.5112 0.3717 C19 0.9258 0.30480.7095 C20 0.4763 0.1572 0.7109 C21 0.1833 0.3270 0.2580 C22 0.19270.4656 0.2359 C23 0.1008 0.2458 0.1993 C24 −0.0043 0.2676 0.1034 C25−0.0708 0.1728 0.0409 C26 −0.1519 0.3799 −0.0163 C27 −0.2252 0.4942−0.0664 S −0.1936 0.2297 −0.0595 N −0.0507 0.3873 0.0719 O1 0.38970.1501 0.2552 O2 0.6748 0.1045 0.3926 O5 0.9464 0.2278 0.4266 O7 0.88930.5485 0.5778 O12 0.3313 0.3359 0.6550 H3 0.5936 0.1283 0.2313 H6 0.74660.4426 0.3937 H7 0.9913 0.3942 0.5683 H8 0.8117 0.2471 0.5514 H13 0.26180.1794 0.5410 H15 0.2633 0.1778 0.3662 H3O 0.6691 0.0154 0.3705 H7O0.9636 0.5994 0.5825 N27 0.4609 0.5049 0.0188 (acetonitrile) C28 0.39630.4080 0.0038 (acetonitrile) C29 0.3379 0.2975 −0.0775 (acetonitrile)

TABLE 3 Fractional Atomic Coordinates for Epothilone B, Ethyl AcetateSolvate, Form EpoB-EAβ (most hydrogen atoms have been omitted) Atom X YZ C1 0.4400 0.2438 0.3107 C2 0.5605 0.2943 0.3190 C3 0.6416 0.19040.3056 C4 0.7559 0.2327 0.2857 C5 0.8532 0.2913 0.3822 C6 0.8410 0.42280.4212 C7 0.8957 0.4275 0.5404 C8 0.8319 0.3405 0.5928 C9 0.7164 0.40000.5966 C10 0.6298 0.3042 0.6134 C11 0.5231 0.3717 0.6266 C12 0.42660.2844 0.6321 C13 0.3066 0.2780 0.5503 C14 0.2581 0.3526 0.4526 C150.2706 0.2867 0.3589 O16 0.3940 0.3148 0.3668 C16 0.7203 0.3272 0.1940C17 0.8079 0.1121 0.2559 C18 0.9092 0.5160 0.3757 C19 0.9227 0.30990.7056 C20 0.4667 0.1703 0.7040 C21 0.1800 0.3335 0.2576 C22 0.18870.4688 0.2331 C23 0.0962 0.2506 0.2011 C24 −0.0076 0.2687 0.1042 C25−0.0762 0.1706 0.0472 C26 −0.1515 0.3743 −0.0242 C27 −0.2158 0.4821−0.0821 S −0.1923 0.2232 −0.0566 N −0.0487 0.3847 0.0652 O1 0.38780.1559 0.2575 O3 0.6749 0.1137 0.3972 O6 0.9464 0.2337 0.4280 O7 0.88890.5526 0.5755 O12 0.3257 0.3484 0.6457 H3 0.5927 0.1346 0.2372 H6 0.74630.4478 0.3947 H7 0.9884 0.3992 0.5620 H8 0.8080 0.2533 0.5499 H13 0.25730.1911 0.5357 H15 0.2575 0.1863 0.3624 H3O 0.6713 0.0150 0.3759 H7O0.9745 0.5994 0.5930 O28 0.5242 0.5794 0.0077 (ethyl acetate) O31 0.41790.4326 0.0063 (ethyl acetate) C28 0.4731 0.5098 0.0256 (ethyl acetate)C29 0.4265 0.4705 0.0892 (ethyl acetate) C31 0.3610 0.3621 −0.0408(ethyl acetate) C30 0.2548 0.3272 −0.0460 (ethyl acetate)

TABLE 4 Fractional Atomic Coordinates for Epothilone B, IsopropylAlcohol Solvate, Form EpoB-IPβ (most hydrogen atoms have been omitted)Atom X Y Z C1 0.4418 0.2548 0.3104 C2 0.5609 0.3055 0.3186 C3 0.64290.2009 0.3049 C4 0.7565 0.2462 0.2851 C5 0.8541 0.3018 0.3837 C6 0.84100.4357 0.4229 C7 0.8977 0.4390 0.5415 C8 0.8345 0.3493 0.5940 C9 0.71840.4107 0.5972 C10 0.6325 0.3111 0.6156 C11 0.5261 0.3793 0.6303 C120.4292 0.2892 0.6329 C13 0.3087 0.2842 0.5528 C14 0.2607 0.3630 0.4551C15 0.2736 0.2932 0.3613 O16 0.3950 0.3241 0.3669 C16 0.7179 0.33800.1935 C17 0.8084 0.1250 0.2541 C18 0.9098 0.5290 0.3774 C19 0.92690.3222 0.7069 C20 0.4742 0.1736 0.7031 C21 0.1807 0.3387 0.2590 C220.1879 0.4780 0.2352 C23 0.1028 0.2530 0.2009 C24 −0.0041 0.2724 0.1029C25 −0.0678 0.1712 0.0456 C26 −0.1517 0.3781 −0.0198 C27 −0.2289 0.4888−0.0775 S −0.1896 0.2262 −0.0575 N −0.0526 0.3893 0.0653 O1 0.39030.1657 0.2594 O3 0.6763 0.1239 0.3954 O5 0.9485 0.2459 0.4293 O7 0.88980.5642 0.5781 O12 0.3283 0.3539 0.6476 H3 0.5946 0.1457 0.2365 H6 0.74640.4597 0.3977 H7 0.9915 0.4115 0.5668 H8 0.8111 0.2625 0.5504 H13 0.25820.1971 0.5380 H15 0.2640 0.1927 0.3679 H3O 0.6731 0.0260 0.3733 H7O0.9599 0.6223 0.5696 O28 0.4344 0.2122 0.0495 (isopropyl alcohol) C280.3601 0.2863 −0.0462 (isopropyl alcohol) C30 0.4351 0.3798 −0.0762(isopropyl alcohol) C29 0.2460 0.3279 −0.0487 (isopropyl alcohol)

TABLE 5 Fractional Atomic Coordinates for Epothilone B, Toluene Solvate,Form EpoB-TOβ (most hydrogen atoms have been omitted) Atom X Y Z C10.4314 0.2211 0.3158 C2 0.5581 0.2739 0.3228 C3 0.6395 0.1704 0.3110 C40.7506 0.2081 0.2888 C5 0.8509 0.2746 0.3880 C6 0.8414 0.4043 0.4212 C70.8976 0.4053 0.5382 C8 0.8372 0.3234 0.5911 C9 0.7204 0.3812 0.5930 C100.6312 0.2790 0.6075 C11 0.5255 0.3494 0.6227 C12 0.4302 0.2588 0.6250C13 0.3014 0.2537 0.5473 C14 0.2538 0.3361 0.4501 C15 0.2643 0.26400.3626 O16 0.3877 0.2964 0.3648 C16 0.7158 0.3123 0.2026 C17 0.80820.0907 0.2610 C18 0.9061 0.4961 0.3806 C19 0.9323 0.2951 0.6989 C200.4703 0.1447 0.6945 C21 0.1702 0.3170 0.2598 C22 0.1709 0.4486 0.2391C23 0.0898 0.2230 0.2030 C24 −0.0145 0.2462 0.1060 C25 −0.0811 0.14300.0546 C26 −0.1432 0.3561 −0.0251 C27 −0.2089 0.4563 −0.0926 S −0.19870.1985 −0.0555 N −0.0507 0.3632 0.0580 O1 0.3838 0.1303 0.2676 O3 0.67420.0956 0.3983 O5 0.9468 0.2169 0.4313 O7 0.8912 0.5329 0.5727 O12 0.32540.3255 0.6408 H3 0.5816 0.1062 0.2496 H6 0.7457 0.4264 0.3944 H7 0.99190.3719 0.5628 H8 0.8141 0.2297 0.5521 H13 0.2871 0.1529 0.5221 H150.2567 0.1589 0.3722 H3O 0.6633 −0.0002 0.3785 H7O 0.9663 0.5756 0.5776C28 0.4258 0.4317 0.0030 (toluene) C29 0.3526 0.3996 0.0429 (toluene)C30 0.2586 0.3239 0.0126 (toluene) C31 0.2245 0.2386 −0.0713 (toluene)C32 0.2984 0.2800 −0.1182 (toluene) C33 0.3923 0.3496 −0.1016 (toluene)C34 0.5043 0.4979 −0.0119 (toluene)

TABLE 6 PXRD Data for Epothilone B, Toluene Containing Solvate, ProducedUsing the Method of Example 7, Step A and Shown in FIG. 9 ScatteringRelative angle d-spacing Intensity (deg. 2-theta) (A) (%) 6.680 13.221248.5 8.210 10.7604 2.1 10.610 8.3312 2.2 12.590 7.0251 4.3 13.370 6.616925.8 14.840 5.9646 1.9 15.680 5.6469 0.9 16.160 5.4802 2.3 16.550 5.35202.9 18.170 4.8783 5.0 18.410 4.8152 10.6 20.090 4.4162 100.0 20.7204.2833 4.2 21.320 4.1641 1.4 21.890 4.0570 6.3 24.200 3.6747 3.2 24.5003.6304 4.7 24.800 3.5871 14.4 26.150 3.4049 4.4 26.870 3.3153 4.5 28.3703.1433 4.3 29.930 2.9829 2.2 30.890 2.8924 2.0 31.400 2.8466 2.2

TABLE 7 PXRD Data for Epothilone B, Toluene Containing Solvate, ProducedUsing the Method of Example 7, Step B, and Shown in FIG. 11 ScatteringRelative angle d-spacing Intensity (deg. 2-theta) (A) (%) 6.680 13.221262.1 8.210 10.7604 3.4 10.610 8.3312 6.9 12.590 7.0251 8.1 13.370 6.616926.9 14.870 5.9526 5.9 15.680 5.6469 3.7 16.160 5.4802 4.5 16.580 5.34249.1 18.170 4.8783 20.3 18.440 4.8075 28.2 20.090 4.4162 100.0 20.7204.2833 9.0 21.320 4.1641 4.4 21.890 4.0570 21.8 24.200 3.6747 10.824.500 3.6304 9.7 24.800 3.5871 18.9 26.150 3.4049 12.8 26.900 3.31174.9 28.340 3.1466 7.6 29.960 2.9800 5.9 30.950 2.8869 5.3 31.400 2.84664.1

TABLE 8 PXRD Data for Epothilone B, Ethyl Acetate Containing Solvate,Produced Using the Method of Example 7, Step C, and Shown in FIG. 13Scattering Relative angle d-spacing Intensity (deg. 2-theta) (A) (%)6.800 12.9881 26.2 6.950 12.7082 32.5 8.450 10.4553 4.0 10.850 8.14749.4 11.690 7.5638 2.8 13.070 6.7681 11.6 13.850 6.3887 10.2 14.9905.9053 4.7 16.040 5.5210 5.4 16.850 5.2574 11.0 18.200 4.8703 13.518.770 4.7237 16.9 19.130 4.6356 14.3 20.480 4.3330 72.2 20.600 4.308071.6 20.720 4.2833 100.0 22.610 3.9294 25.5 24.470 3.6347 8.9 24.8603.5786 13.5 25.190 3.5325 7.8 26.120 3.4088 5.7 26.600 3.3483 5.9 27.0503.2936 3.5 27.530 3.2373 5.2 28.850 3.0921 6.7 29.090 3.0671 6.7 30.0202.9742 4.0 30.320 2.9454 4.6 30.740 2.9062 5.6 31.220 2.8626 6.5

DEFINITIONS

The following terms shall have, for the purposes of this application,the respective meanings set forth below:

“Epothilone B comparative production conditions.” To measure relativeproduction of epothilone B to epothilone A, or net epothilone Bproduction between strains, standard conditions are needed. The“epothilone B comparative production conditions” are set forth below.Note that standard conditions may be appropriately scaled (e.g., to 125mL production flasks according to Example 2) as described in theExamples:

1) F1 Stage:

One mL from a frozen vial or maintenance flask is transferred to a 125mL flask containing ca. 10 mL Medium E (composition described below).The F1 flask is incubated for 3–4 days at 30° C. and 160 rpm.

2) F2 Stage:

The entire contents from the F1 flask (ca. 10 mL) are transferred (10%)to a 250 mL flask containing 90 mL Medium E. This F2 flask is similarlyincubated for 3–4 days at 30° C. and 160 rpm.

3) Production Stage:

Production flasks (250 mL flasks containing 90 mL Medium E, see mediumformulations below) are inoculated at a level of 10% (10 mL) from the F2stage. Alternatively, “maintenance flasks” may be used, and these arederived from routine flask transfer of culture every 3–4 days at levelsranging from 5% to 10%. The production phase incorporates at least 15g/L of resin. Once inoculated, production flasks are incubated at 30° C.and 160 rpm for 14 days. A feed is incorporated to improve theepothilone B to A ratio. Feed additions begin at 72 hourspost-inoculation as follows:

One mL of feed is added per production flask (100 mL culture volume) perday from days 3–11 with additions also continuing through day 14 whereindicated.

The propionate-containing feed contains 10% Maltrin-M040, 4% sodiumpropionate, and 3% Tastone-154, such that when added as described at a100-fold dilution, the final concentration in the culture broth, perday, becomes 0.1% Maltrin-M040, 0.04% sodium propionate, and 0.03%Tastone-154 (excluding residual levels from prior additions). Flaskswere generally harvested for assay 14 days post-inoculation.

“Propionic acid precursor” refers to any compound that can be added toan appropriate culture in an amount effective to generate an amount ofpropionic acid effective to increase the epothilone B to epothilone Aratio. Propionic acid can be generated spontaneously, for example, withlabile esters through the action of cellular enzymes. Those of ordinaryskill in the art shall recognize candidate compounds which can bereadily tested for generating propionic acid or for increasing theepothilone B to epothilone A ratio. Examples include methyl and ethylesters of propionic acid.

By “feeding,” it is meant that at least one or more nutrients oradditives, such as propionate, sodium propionate, a sodium propionatecontaining mixture or solution, a vitamin, a mineral, a carbohydratesource or an amino acid source, is added on more than one occasionduring the course of the fermentation, such as, for example,periodically, via a pulse feed, via a substantially continuous feed, andthe like. It should be understood that a continuous feed throughout thefermentation is included within the meaning of the term “added on morethan one occasion.”

“Toluene-containing” means a solvate predominantly containing an amountof toluene as measured by analytical techniques used by those skilled inthe art, wherein the toluene-containing solvate may or may not alsocontain one or more additional solvents.

“Ethyl acetate-containing” means a solvate predominantly containing anamount of ethyl acetate as measured by analytical techniques used bythose skilled in the art, wherein the ethyl acetate-containing solvatemay or may not also contain one or more additional solvents.

EXAMPLES

The examples below are carried out using standard techniques that arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the present invention.

Example 1 Preparation of the Strain SC16408 by Means of Mutation andSelection, and Preparation of Cell Banks

Strain SC16408 was derived from the nitrosoguanidine (NTG) treatment ofstrain So ce90B2 (SC16224), followed by random selection. Thus, SC16224was suspended in 10 mM Tris-HCl buffer and subjected to 1 mg/mL NTG for60 minutes at pH 8.2. After treatment with NTG, colony cell lines wereobtained by colony selection and tested for epothilone B productivity,and B/A ratio. Isolated colonies were transferred to flasks and culturedfor 8–14 days, followed by transfers every 3–4 days in the growth medium(medium E):

Growth Medium E for shake flasks:

Ingredient g/L Powdered Skim Milk 4 Toasted Nutrisoy Flour 4 Tastone-1542 Maltrin-M180 10 CaCl₂.2H₂O 1 MgSO₄.7H₂O 1 EDTA, FeIII, Na salt 0.008HEPES 12 Glycerol 4.3The above ingredients are added to distilled water and the pH isadjusted to pH 7.2 with 10% NaOH (or KOH) before sterilization for 30minutes at 121° C.

Preparation of research cell bank: a volume of 10 mL from a 3-day oldculture of strain SC16408 was transferred into a 250 mL flask containing90 mL of medium E. The flask was then incubated at 30° C., 160 rpm for 2days. At the end of 2 days, 1.8 mL aliquots were withdrawn from theflask and transferred into cryogenic vials, which were then frozen at−70° C.

Preparation of master cell bank: 2 vials from the research cell bankwere thawed and transferred into 2×125 mL flasks containing 10 mL ofmedium E, and then incubated at 30° C., 160 rpm for 4–5 days. Next, 2×10mL were transferred into 2×250 mL flasks containing 90 mL medium E andincubated at 30° C., 160 rpm for 2–4 days. Finally, these 2 flasks werepooled and 1.8 mL aliquots were transferred into cryogenic vials andstored in a freezer at −70° C.

Preparation of working cell bank: 5 vials from the master cell bank werethawed and transferred into 5×125 mL flasks containing 10 mL of mediumE, then incubated at 30° C., 160 rpm for 3–6 days. Next, 5×10 mL weretransferred into 5×250 mL flasks containing 90 mL medium E and incubatedat 30° C., 160 rpm for 2–4 days. Cells in these 5 flasks were used toinoculate 12×250 mL flasks containing 90 mL medium E which were againincubated at 30° C., 160 rpm for 2–4 days. Finally, these flasks werepooled together and 1.8 mL aliquots were transferred into cryogenicvials and stored in a freezer at −70° C. About 500–600 vials weregenerated for this working cell bank.

Example 2 Cultivation to Produce the Epothilones by Shake FlaskFermentation

Cells from a frozen vial (1.5 mL) are inoculated into 45 mL of medium Ein a 125 mL flask and grown for 4–8 days at 30° C. and 160 rpm (F1stage). Then, 5 mL of the F1 stage are transferred to a new 125 mL flaskcontaining 45 mL medium E and grown for 3–4 days (F2 stage). F2 stagecells are then used as inoculum for epothilone B fermentations. Tenpercent of inoculum (5.0 mL) is transferred into a 125 mL flaskcontaining 45 mL production medium. The flasks are then incubated in ashaker (160 rpm) at 30° C. for 2 weeks. The production medium ismodified medium E, which contains 1.6% (0.8 g) XAD-16 resin. Thecomposition of the production medium for shake flasks is shown:

Epothilone B production medium for shake flasks:

Ingredient g/L Powdered Skim Milk 4 Toasted Nutrisoy Flour 4 Tastone-1542 Maltrin-M040 10 CaCl₂.2H₂0 1 MgSO₄.7H₂0 1 EDTA, FeIII, Na salt 0.008HEPES 12 Glycerol 10 XAD-16 resin 16The above ingredients are dissolved in distilled water and the pH isadjusted to 7.2 with 10% NaOH (or KOH) before sterilization for 30minutes at 121° C.

The composition of feed solution for the shake flask epothilone Bfermentation is: 4% sodium propionate, 10% Maltrin-M040 and 3%Tastone-154. The feed (100 mL in a 250 mL flask) is adjusted to pH6.8–7.0 with NaOH and sterilized for 30 minutes at 121° C. From day 3 today 14 post inoculation, 0.5 mL of feed solution is added daily to eachfermentation flask. Alternatively, it has been found that comparableresults may be achieved by doubling the feed levels and performing theadditions at days 3, 5, 7 and 10. Improved results can also be achievedby further supplementing the above feed solution with phosphate, in theform of 1.5% dibasic sodium phosphate and 0.5% monobasic sodiumphosphate, such that when diluted 100-fold into the culture medium,final levels are 0.015% and 0.005%, respectively, excluding residuallevels from prior additions. An added advantage of phosphate addition isthat no pH adjustment needs to be performed. Additional yieldimprovements (in epothilone B) as high as 10–20% can be achieved throughphosphate supplementation.

For assay of epothilones, resin samples (0.8 g) are harvested andassayed by HPLC. Epothilone production in shake flasks should yield thefollowing titers at 14 days:

-   -   Epothilone A: 5.0–7.0 mg/g resin    -   Epothilone B: 8.0–12.0 mg/g resin    -   B/A ratio: 1.1–2.0

Compared to previous strains, the SC16408 culture appears to producemore epothilone B in shake flasks.

Example 3

Cultivation to produce the epothilones in 14 L fermentors F1 stage: a3.0 mL aliquot from two frozen vials is inoculated into 90 mL of mediumE in a 250 mL flask and grown for 4–8 days at 30° C. and 160 rpm. F2stage: 20 mL (10%) F1 stage cells are transferred to 180 mL of medium Ein a 500 mL flask and incubated for 2–4 days at 30° C. and 160 rpm. F3stage: Repeat F2 stage to increase inoculum quantity. Transfer 20 mL ofinoculum from F2 stage into 6–8 × 500 mL flasks each containing 180 mLof medium E, and incubate flasks for 2–4 days at 30° C. and 160 rpm. F4stage: Transfer 120 mL (10%) from F3 stage to 1080 mL of medium E in a 4L aspirator bottle, then incubate for 2–4 days at 30° C. and 160 rpm.

Medium E is used to build up the inoculum for a 14 L fermentor. Theautoclave times for shake-flask and aspirator-bottle stages are 30 and60 minutes, respectively. For the fermentor, the production medium issterilized for 60 minutes at 121° C. The 14 L fermentor productionmedium is a modified shake flask production medium (as described above)where HEPES has been deleted and 2.5 g/L of an antifoam agent (AntifoamAF, from Dow Coming) has been added. Six liters of production medium (pHadjusted to 7.2–7.4) is dispensed in a 14 L fermentor and sterilized.The table below summarizes the process parameters at the 14 L fermentorscale:

Bench top fermentor process parameters:

F1 to F4 14 L Temperature 30° C. 32° C. Pressure 10 psi Airflow 0.25 vvmpH 7.2–7.4 DO  20–40% Impeller diameter (in) 3.3–4.2 Tip speed (m/s)1.3–2.2 Feed sterilization time 60 min Media sterilization 30 min 60 mintime Resin  15–30 g/L

Nutrient feed composition: A solution composed of 4.1% Maltrin- M040 and1.3% Tastone-154 is prepared in a 5 L bottle. The feed is sterilized for60 minutes at 121° C. Nutrient feed rate: The feed rate is 6 mL/hour.Sodium propionate feed: 5.0% sodium propionate (1.5 L in 2 L bottle) issterilized for 60 minutes at 121° C. Sodium propionate feed rate: From24–48 hours to finish, 2 mL/hour. The feed rate is adjusted to maintainsodium propionate concentration between 0.05–0.2 mg/mL based on HPLCassay.The epothilone B titer range in 14 L fermentors is summarized below:

Epothilone B titer, mg/g resin B/A Ratio 5–12 1.0–3.0

Example 4 Manufacturing Process for Epothilones

50 L Fermentor Seed Stage:

For the F1 stage, medium E (2 L) is made up and dispensed, 90 mL eachinto 17 separate 250 mL flasks. The flasks are then sterilized byautoclaving at 121° C. for 30 minutes. Cells from one frozen vial areinoculated into each flask and grown for 4–8 days at about 30° C. and160 rpm.

For the F2 stage, 27 L of medium E are made up and dispensed, 1.5 L eachinto 17 separate 4 L flasks, then sterilized as above. Each 4 L flask isinoculated with the entire contents of a flask from the F1 stage, thengrown for 2–4 days at about 30° C. and 160 rpm.

For the F3 stage, 80 L of medium E* is made up and divided into two 50 Lstainless steel seed fermentors and each 50 L fermentor is inoculatedwith the contents of three 4 L flasks from the F2 stage. The 50 Lfermentors are grown for 2–4 days at 30–33° C., then combined and usedto inoculate an 800 L fermentor.

Medium E* is:

Ingredient g/L Powdered Skim Milk 5 (or soy protein concentrate) ToastedNutrisoy Flour 5 Tastone-154 2.5 Maltrin-M040 12.3 CaCl₂.2H₂O 1.2MgSO₄.7H₂O 1.2 EDTA, FeIII, Na salt 0.012 Glycerol 5.4 Antifoam 2.5800 L Fermentor Seed Stage:

The inoculum is grown in an 800 L stainless steel fermentor until thecell mass is sufficient to inoculate the next seed stage (a 5,000 Lfermentor).

Medium E* for the batch is made up into deionized water (400 L) and themixture, pH 8.7–8.9, is sterilized at 17 psig, 124° C. for 60 minutes.The medium is transferred from the sterilizer to the 800 L fermentor,and pH adjusted to pH 7.1–7.3. The fermentor is then inoculated with 80L from the F3 stage. The batch is run with the following control setpoints:

Pressure:  8–12 psig Air Flow: 0.5–0.7 vvm Temperature: 30–33° C. pH:7.1–7.3 Agitator shaft speed: 50–60 rpmAs needed, caustic (sodium or potassium hydroxide solution) is addedfrom a sterile supply to maintain pH in the 7.1–7.3 range. The batch issampled at intervals and analyzed for sterility, pH, sediment andglucose concentration. Vent off-gas CO₂ and O₂ are also monitored. Atapproximately 48–60 hours, when the glucose concentration is starting tofall, the contents of the 800 L fermentor (approximately 440–480 L) aretransferred to a 5,000 L fermentor.5,000 L Fermentor Seed Stage:

A 5,000 L stainless steel fermentor is used in the inoculum process atthis stage. The inoculum is grown in the fermentor until the cell massis sufficient to inoculate the 40,000 L production fermentor.

Medium E*, prepared as above (into deionized water, 2,600 L), istransferred to the 5,000 L fermentor and then inoculated withapproximately 440–480 L of inoculum from the 800 L fermentor. The batchis run with the control set points and monitoring described above.Again, pH is maintained in the 7.1–7.3 range. At about 48–72 hours, whenthe glucose concentration begins to fall, the contents of the 5,000 Lfermentor are transferred to the 40,000 L fermentor.

40,000 L Fermentor Production Stage:

A 40,000 L stainless steel fermentor is used in the production of theepothilones. Once the fermentor has been sterilized and filled withsterile medium, it is inoculated with the seed prepared in the 5,000 Lfermentor. Once specific production parameters are achieved, thecontents of the production fermentor are harvested.

The medium for the production fermentor is sterilized in two parts. Theresin is added into 2,800 L of water and the mixture is sterilized at 17psig, 124° C. for 75 minutes:

Ingredient Amount Washed XAD-16 Resin 15–40 g/L

To make 18,000 L of medium, the following ingredients are added intodeionized water (15,000 L) and the pH is adjusted to 7.1–7.3. The mediumis sterilized at 150° C. in a continuous sterilizer (hold time 100seconds, outlet temperature 60° C.):

Ingredient Weight (kg) Powdered Skim Milk 130 Toasted Nutrisoy Flour 130Tastone-154 65 Maltrin-M040 238 CaCl₂.2H₂O 21.6 MgSO₄.7H₂O 21.6 EDTA,FeIII, Na salt 0.22 Glycerol 216 Antifoam 54

The medium and resin are transferred to the production fermentor whichis then inoculated with approximately 3,100 L of inoculum from the 5,000L fermentor. The batch is run with the control set points describedabove, except air flow is 0.2–0.4 vvm. As needed, the pH (between 0 and80 hours) is raised with caustic. After 80 hours, the pH is lowered withsulfuric acid. As needed, foaming is controlled with antifoam. Thefermentor is sampled at least once a day for sterility, pH, sediment,glucose, propionate and epothilone B concentration. CO₂ in the off-gasis monitored and recorded. Feeds are started at approximately 30–60hours, as long as the CO₂ is at least 0.3%.

The fermentor is fed sodium propionate (102 g/L) with a shot size of 1.9L/shot (range 1.5–3.0). The interval between shots starts at 60 minutesand decreases every 12 hours to a minimum of 12 minutes. The propionateis added into 2,800 L of deionized water and the solution is sterilizedat 17 psig, 124° C. for 75 minutes. In a preferred embodiment, thesodium propionate feed is separate from the feed containing other mediacomponents.

The fermentor is fed Maltrin-M040 and Tastone-154 with a shot size of14.5 L. The interval between shots starts at 60 minutes and changes at104 hours to 40 minutes. The ingredients are added to deionized water(3,000 L) and sterilized at 17 psig, 124° C. for 75 minutes. The feedcomprises:

Ingredient g/L Tastone-154 20 Maltrin-M040 66 Antifoam 1.0

During the run, some of the medium components such as powdered skimmilk, Maltrin-M040 and glycerol are exhausted. Starting at approximately115 hours, the previous feed is discontinued and the following mixturewith a shot size of 14.5 L is added to the production fermentor atintervals of 40 minutes. The ingredients are added into deionized water(3,000 L) and the mixture, pH 8.7–8.9, is sterilized at 17 psig, 124° C.for 75 minutes:

Ingredient g/L Powdered Skim Milk 49 Maltrin-M040 154 Tastone-154 20Glycerol 78 Antifoam 1.6When a desired epothilone B concentration is achieved (normally after9–21 days), the contents of the vessel are harvested. Epothilone Btiters range from approximately 5–24 mg/g resin, with B/A ratios fromapproximately 1.5–4.

Example 5 Extraction of Epothilone B from XAD-16 Resin with MTBE andCrystallization to Give Solid Epothilone B; Purification by ReversePhase Chromatography; and Final Isolation of High-quality Epothilone B

Harvested and water-washed XAD-16 resin (approximately 550 kg)containing epothilones (approximately 5.03 kg epothilone B) is mixedwith aqueous methanol and loaded into an extraction column as a slurry.The packed resin is washed with aqueous methanol (1 bed volume each of30% then 50% MeOH) to remove highly polar undesired materials.Epothilones are removed with MTBE washes (approximately 4 bed volumes).The rich eluate is collected and polish filtered. After gravity settlingto remove any aqueous phase, the rich MTBE is concentrated. Theconcentrate is gravity settled, the aqueous phase removed, andadditional MTBE (2 bed volumes) added to the batch. The batch isre-concentrated to a concentration of approximately 5 to 15 g epothiloneB per L. The batch is crystallized by gradual cooling over 5–6 hours atapproximately 0° C. The crystalline solid is filtered, washed and dried.The resulting product cake is dissolved in warm ethyl acetate and polishfiltered. The rich filtrate is concentrated under vacuum to aconcentration of approximately 20 to 45 g epothilone B per L. Afterheating to 70° C., the batch is then cooled slowly to approximately 0°C. to give a crystalline slurry which is filtered, washed with coldEtOAc, and dried at less than 40° C. to give isolated recrystallizedepothilone B (in 84% yield from the resin). This product is thenpurified by reverse phase chromatography.

A chromatographic column (11 cm diameter×40 cm bed length) packed withreverse phase stationary support RP/C-18 is equilibrated with aqueousacetonitrile (30–50% v/v). Recrystallized product is dissolved indimethyl sulfoxide (DMSO, 1–1.5 L per kg), the mixture filtered toremove insoluble materials, then loaded onto the column preceded by analiquot of 100% DMSO, and chased by an equal volume of DMSO to reduceprecipitation of the sample upon introduction of the aqueous mobilephase. The sample is eluted from the column using aqueous acetonitrile(30–50% v/v), and the effluent is monitored at 290 nm by a UV detector.The epothilone B product peak is collected in a number of fractions. Thefractions are assayed by HPLC for both epothilone A and B and otherrelated impurities.

Desired pooled column fractions are charged to a distillation package,and the batch vacuum-concentrated to remove the acetonitrile at atemperature below 40° C. The resulting aqueous phase is extracted up tothree times with ethyl acetate, and the organic solution is concentratedunder vacuum at a temperature below 40° C. to give a concentration of0.1 to 0.2 g/mL of epothilone B. n-Heptane (or heptanes) is added to thebatch at 40° C., then the batch is cooled slowly to 2 to −10° C. andheld for at least 2 hours. The crystal slurry is filtered and washedwith an ethyl acetate/n-heptane solution, then the final epothilone Bcake is dried under vacuum at 35–40° C. to yield 3.367 kg with a potencyof 91.7% equivalent to 3.09 kg of epothilone B activity. The yield fromthe resin was 61.4%. HPLC indicated 99.6 area % epothilone B, 0.4 area %epothilone A, with no other impurity present at >0.1 area %.

Example 6 Extraction of Epothilone B from XAD-16 Resin with EthylAcetate and Crystallization with Toluene as Antisolvent to Give SolidEpothilone B; Purification by Reverse-Phase Chromatography; FinalIsolation of High-quality Epothilone B

XAD-16 resin containing epothilone B is washed with water on a vibratingscreen (SWECO TM) to clean the resin. A portion of this (approximately6.6 L, containing 15.6 g epothilone B, assay 2.36 mg of epothilone B pergram of resin) is transferred to a 20 L container using approximately 5L of water to rinse the resin with water. Ethyl acetate (approximately 2bed volumes (BV) of input resin) is then added to the container. Theslurry is stirred for about one hour, and centrifuged at 3,500 rpm for 5minutes using 600 mL screw cap centrifuge jars to separate layers. Thefirst rich ethyl acetate supernatants are decanted, and their volumesare measured. Next, the lean aqueous resin-containing bottom layers arepooled in the container, and ethyl acetate(2 BV) is added to thecontainer. The slurry is stirred for about 1 hour and then centrifugedto separate layers. The second rich ethyl acetate supernatants aredecanted, and their volumes are measured.

Water (˜0.3 BV of input resin) is then added to the combined rich firstand second ethyl acetate streams and agitated for approximately 5minutes. Layers are permitted to settle for approximately 30 minutes.Next, the lower aqueous layer is separated from the upper rich ethylacetate layer. The rich washed ethyl acetate layer is concentrated at atemperature less than 45° C., to a concentration of approximately 10 gof epothilone B activity per liter. The concentrated rich ethyl acetatesolution is then polish filtered and concentrated to 20–25 g/L ofepothilone B.

After concentration, toluene is added and the batch is re-concentratedusing vacuum at less than 50° C., to the volume of the batch beforetoluene addition. The batch is allowed to cool to about 18° C. overapproximately 1 hour, then stirred for approximately 16 hours at thistemperature to produce product crystals. Next, the crystallization batchis filtered and washed with toluene (˜0.2 BV) and the solids are driedto yield approximately 30.4 g of solid containing 13.5 g of epothilone Bactivity. The activity yield from starting resin is 87%.

Purification by reverse-phase chromatography is performed on the solidepothilone B extracted from resin using the above process. The column(Phenomenex Luna, 15, C18(2), 5.0 cm×25 cm, column BV 400 mL) ispre-equilibrated with 3 BV of 40% (v/v) acetonitrile-water.Approximately 4–6 g of the solid epothilone B is dissolved inapproximately 6 mL of DMSO at about 40° C., then the mixture is filteredthrough filter paper to remove particulates. Approximately 1.5 mL ofDMSO is injected into the sample loop to prevent precipitation ofepothilones in the tubing. The epothilone-rich filtrate is then injectedinto the sample loop. Following the injection, the epothilone filtratecontainer is washed with around 0.5 mL of DMSO and injected along withabout 1 mL of DMSO into the sample loop. Injection of DMSO afterinjection of the epothilone sample prevents precipitation in the tubing.The contents of the sample loop are loaded onto the column at a flowrate of approximately 5 mL/min.

After loading the epothilone onto the column, the 40% acetonitrile-watersolution is then pumped through the column. After 3–4 minutes, the flowrate is increased to approximately 60 mL/min. The epothilone A and Bpeaks are collected in fractions. The rich epothilone B-containingfractions typically are obtained in the cuts between about 2.5 L and3.25 L eluted volume (the epothilone B peak typically elutes betweenabout 6 and 8 bed volumes). The volume of the pooled fractions isapproximately 0.75 L. After the peak for epothilone B has nearly reachedbaseline (<10% of peak height), 100% acetonitrile is pumped through thecolumn. When the chromatogram indicates that the absorbance at 290 nmhas essentially returned to baseline, re-equilibration of the column isinitiated for the next run by pumping 40% acetonitrile-water solutiononto the column. Typically 2 BV of 100% acetonitrile and 3 BV 40%acetonitrile are used to wash and re-equilibrate the column.

Fractions are assayed to determine purity using HPLC analysis, and thedesired fractions pooled. Typical yields are 90–98%.

The pooled epothilone B fractions are concentrated under vacuum at lessthan 40° C., to approximately 50% of initial volume. The concentratedfractions are extracted with ethyl acetate. The pooled ethyl acetateextracts are concentrated to approximately 0.1 g/mL epothilone B at abath temperature of approximately 40° C. While stirring, n-heptane (orheptanes) (using a volume of 50% of the ethyl acetate solution) is addedover a period of about 15 minutes. Extracts are cooled to 5° C. and heldat that temperature for at least 2 hours. The product crystals arefiltered and washed with a 1:2 (v:v) n-heptane:ethyl acetate solution.Finally, crystals are dried under vacuum at approximately 40° C. forapproximately 12 hours. HPLC indicated, for various batches. 99.5–99.7area % epothilone B, and 0.3–0.5 area % epothilone A.

Example 7 Extraction of epothilon B from XAD-16 Resin with Ethyl Acetateand Crystallization with Toluene as Antisolvent, Followed byRecrystallization to Give Primary Grade Epothilone B; Purification byNormal-phase Chromatography; and Final Isolation of High-qualityEpothilone B

Step A, Preparation of Primary Grade Epothilone B using EtOAcExtraction-toluene Crystallization:

Water washed epothilone B rich resin (1350 g) is loaded onto a column.Water (2700 mL) is used to load and rinse the column. The epothiloneactivity is eluted by passing 9450 mL (7 bed volumes) of ethyl acetatethrough the column. The ethyl acetate eluate is allowed to settle for atleast one hour. A dark brown aqueous layer and an emulsion layer areremoved. The rich ethyl acetate solution is concentrated under vacuum toa target concentration of approximately 20 g of epothilone B per L. Theconcentrate is allowed to stand 2 hours and cooled to 20° C. The cooledconcentrate is polish filtered, and the filter washed with ethyl acetate(36 mL). The combined filtrate and wash are concentrated toapproximately 80 g epothilone B per L and heated to 65° C. An equalvolume of toluene is added with stirring over 10–15 minutes whilekeeping the temperature above 60° C. The temperature is maintained at65° C. for 30 minutes followed by lowering the temperature to 40° C.over 1.5 hours and then lowering the temperature to 1° C. over 2 hours.The resulting crystalline slurry is stirred at 1° C. for at least 60minutes. The solids are filtered off and washed with toluene (20% of theslurry volume). (In various repetitions of this method, the motherliquor typically contains 2–6% of the input epothilone B activity). Thesolids are dried in a vacuum in an oven at 40–45° C. for at least 4hours. Alternatively, the solids are dried in a vacuum in an oven at atemperature between about 40° C. and room temperature for at least 4hours.

The dry primary epothilone B cake weight ranged from 8.4 to 20 g, theepothilone B potency ranging from 650 to 713 μg/mg. The cake alsocontained 12% to 26% epothilone A (area percent). The residual solventlevels were 0.7% (w/w) EtOAc and 13% (w/w) toluene.

For five lots that were evaluated:

Epo B in Weight assay (g epo B input Primary input (g) epo B/Kg) (g)cake (g) % recovery 1327–1391 4.4–12.2 6.0–16.5 5.5–13.6 87–98

The total losses for the isolation process averaged 9.4%. The percent ofthe epothilone A peak relative to the epothilone B peak from resin toprimary cake dropped from an average of 49% to 19%. The PXRD pattern andthermal analysis for crystal solvate obtained following the methoddescribed in this step are set forth in FIGS. 9 and 10, respectively.The PXRD pattern of FIG. 9 is further characterized by the data reportedin Table 6, above.

Step B, Recrystallization of Epothilone B:

EtOAc (0.14 L) is added to 15 g primary grade epothilone B (710 μg/mg)and heated to 65–68° C. with stirring. (The target concentration ofepothilone B was 75–80 g activity per liter). Toluene (0.14 L) is addedover 20 minutes while maintaining a temperature above 60° C. Theresulting slurry is held at 65° C. for 0.25 hours to 1 hour. The batchis then cooled to 40° C. over 3 hours. Cooling of the batch is continuedto 0–2° C. over 2 hours. The batch is then held at 0–2° C. for 12 hours.The resulting crystalline slurry is then filtered and the cake washedwith toluene (2×0.028 L). Typically, less than 3% of the inputepothilone B activity is lost to the combined mother liquor and wash.The cake is dried in a vacuum oven at 42° C. and 29 in. Hg for 2 hours.Alternatively, the cake is dried in a vacuum oven at a temperaturebetween about 40° C. and room temperature and 29 in. Hg for 2 hours. Thedry cake weight is 13.6 g with a potency of 764 μg/mg. Residual solventsinclude EtOAc (0.9 wt %) and toluene (13.2%). Typically, EtOAc andtoluene are present at combined levels of 13–14 wt %. The percent of thearea of the epothilone A peak relative to the epothilone B peak forrecrystallized cake dropped to an average of 6.9%.

The PXRD pattern and thermal analysis for crystal solvate obtainedfollowing the method described in this step are set forth in FIGS. 11and 12, respectively. The PXRD pattern of FIG. 11 is furthercharacterized by the data reported in Table 7, above.

Normal Phase Chromatography:

The following mobile phases are prepared:

-   20% (v/v) ethyl acetate/n-heptane solution (˜10 L),-   40% ethyl acetate/n-heptane (˜10 L), and-   100% ethyl acetate (˜10 L).    The following equipment is set up:-   Waters Delta Prep 4000; Detector: UV set at 290 nm; Column:    Phenomenex Luna, 10 micron, silica (2), 5.0 cm×25 cm (column volume    ˜490 mL).

The column is equilibrated with 3 bed volumes of 20% (v/v) ethylacetate/n-heptane solution prior to injection of the epothilonesolution.

The epothilone B cake (5.5 g) is dissolved in 55 mL of methylenechloride. The batch is filtered through a 1-micron PTFE filter to removeany particulates that may be present. Methylene chloride (2–5 mL) isused to rinse the filter. The rich methylene chloride filtrate isinjected onto the column at an initial flow rate of 5 mL/min for thefirst 30 seconds, followed by increasing the flow to 20 mL/min until thesample is fully loaded. The container containing the epothilone filtrateis rinsed with methylene chloride (2–5 mL), and the rinse is also loadedonto the column.

The elution is begun with 20% EtOAc/heptane while increasing the flowrate to 118 mL/min. After the flow rate reaches 118 mL/min, the pumpprogram controller is used to run the desired pump program. Thefollowing pump program is used:

% ethyl acetate/ heptane Time flow (mL/min) A B C volume mL bed volumesBV 0 118 20 7.5 118 20 897 1.83 7.6 118 40 45.6 118 40 4496 9.18 45.7150 100 69.3 150 100 3555 7.26 69.4 150 20 85.7 150 20 2460 5.02 85.8 020

Fractions are collected and assayed for purity using HPLC.

In five batches, the area percent of epothilone B in the collectedfractions was 99.59–99.93%, with yield averaging 91%.

Optionally, the chromatography is performed similarly using an isocratic40% ethyl acetate/n-heptane elution step, with the similar 100% ethylacetate column washing step followed by re-equilibration with 40% ethylacetate. The same chromatography equipment is used with a smallerdiameter column 1.0 cm×25 cm. The chromatography yield for epothilone B(164 mg) in the heartcut fractions was 86% and the area percent ofepothilone B in the collected fractions was 99.4%. The above isocraticprocess was also performed on an 11 cm axial compressed column with31–35 gm of epo B eluted in the heartcut with chromatographic yields of90–94%. The area percent of epothilone B in the collected heartcuts was99.6–99.9%. The column can be re-used multiple times.

Step C, Final Crystallization:

The desired heart-cut fractions are pooled together to give a batchvolume of 1.62–1.73 L. The solvent is removed under vacuum at 40–45° C.The target distillation volume is 25–28 mL (epothilone B concentrationof approximately 200–210 g/L). To the concentrate is added warm(approximately 40° C.) heptane (50 mL). Alternatively, warm(approximately 40° C.) EtOAc (50 mL) could be added to the concentrateat this step. The resulting slurry is stirred at approximately 40° C.for about 2 hours, then is cooled to approximately 0° C. over about 5hours and is further stirred at approximately 0° C. for a minimum of 5hours. Mechanical stirring at moderate rate is used throughout thecrystallization. The crystalline slurry is filtered and the cake iswashed with cold 1:1 EtOAc/heptane (25 mL). The solids are dried in avacuum oven at 40–45° C. for 5–6 hours. Alternatively, the solids aredried in a vacuum oven at a temperature between about 40° C. and roomtemperature for 5–6 hours. The weight of isolated epothilone B (for fivebatches) is approximately 4.2–5.0 g (83.5–85.6% activity yield) fromrecrystallized (one time) epothilone B, and the HPLC purity is 99.78 to99.93% area percent (average 99.80%). The epothilone B lost in themother liquor is approximately 4% with respect to epothilone B activityinput to chromatography. Residual solvents in the cake are EtOAc(5.8–6.0% w/w) and heptane (0.6–0.7% w/w). The potency of the finalepothilone B cake ranges from 91.5 to 92.7% w/w. The HPLC purity isabove 99.7 area percent. The PXRD pattern and thermal analysis for acrystal solvate obtained following the method described in this step areset forth in FIGS. 13 and 14, respectively. As can be seen in FIG. 14,the melting point for the ethyl acetate solvate prepared and driedaccording to the above procedure is approximately 102° C. The PXRDpattern of FIG. 13 is further characterized by the data reported inTable 8, above.

Alternatively, pooled heart cuts containing 4.83 Kg of Epo B (1790 L)were concentrated under vacuum at <30° C. to a target concentration of200–210 g/L and then n-heptane (60 Kg) was added. This concentration wasrepeated and then an additional 60 Kg of heptane was added. The slurrywas cooled to 20° C. over three hours, then collected and washed with 30Kg of heptane. The solids were dried at 20–36° C. for 16 hours undervacuum. A total of 5.141 Kg of solid were obtained with an HPLC purityof >99.6 area %. Residual solvents in the cake were 10.6% ethyl acetateand 1.4% heptane.

Example 7A Extraction of Epothilone B from Resin, Followed by RepeatedRecrystallization

Water washed epothilone rich resin (549.8 kg) containing an estimated4.10 Kg of epothilone B activity (Area % for epothilone B=58.0%;epothilone A=29.2%) was slurried with water and charged to a column (700L). The column was drained and blown with nitrogen. Ethyl acetate (2969Kg) was then eluted through the column at a rate of ˜1 bed volume perhour for a total of ˜6 bed volumes. The combined rich ethyl acetateeluate was allowed to gravity settle for ˜1 hour before removing thelower aqueous phase. The rich ethyl acetate was then concentrated to˜574 kg. The concentrated rich ethyl acetate was then allowed to standat ˜20° C. for ˜2 days before polish filtering. The filter and lineswere washed with ethyl acetate (˜115 kg total). The polish filtrate andwash was then concentrated to a volume of ˜64 L, then warmed to ˜65° C.An equal volume of warm toluene was then added with stirring and thematerial was held at ˜65° C. for ˜30 minutes. The batch was then slowlycooled to ˜40° C. over ˜4 hours, followed by cooling to 0° C. over ˜2.5hours. The cold slurry was then held at ˜0° C. for ˜1 hour. Theresulting crystalline slurry was then filtered and the cake washed withtoluene. (˜64 L). The resulting cake was dried briefly under vacuum thenredissolved from the filter dryer using warm ethyl acetate (˜200 kg).

The first recrystallization was performed similarly by concentrating therich ethyl acetate to ˜65 L. After warming to 65° C. an equal volume oftoluene was added with stirring and the material was held at ˜65° C. for˜30 minutes. The batch was cooled similarly as above and the resultingcrystalline slurry was filtered and washed using the same procedure andequipment as above.

Two additional similar recrystallizations as described above wereperformed to yield an epothilone B crystalline cake with (4.384Kg)(81.5% w/w) (3.573 kg epothilone B) (HPLC Area %'s for: epothiloneB=97.18: epothilone A=1.40; epothilone F=0.30; oxazole analog=0.30 andethyl thiazole analog=0.56). No other impurities were detectable by HPLCover 0.1 area percent. The product contained 13.8% w/w toluene and 0.8%w/w ethyl acetate. The overall activity yield from resin to isolatedpurified epothilone B was 87%.

Example 7B Recovery of Epo B from Mother Liquor Streams

Mother liquors from the crystallization of Epo B from MTBE or EtOAcextracts were combined and contained 2.2 g of Epo B and 4.8 g of Epo Aper liter of solution. Ten liters of this solution were concentratedunder vacuum at ≦50° C. to a concentration of 11–15 g of Epo B perliter. One volume of toluene was added and solids began forming;distillation was continued until a concentration of 11–15 g Epo B/L wasagain achieved. One volume of toluene was added again and thedistillation was repeated once more to reach 11–15 g Epo B/L. The slurrywas cooled to room temperature over 1 hour, then stirred for 90 minutes.The mixture was then re-heated to ˜50° C., stirred for 1 hour and cooledto room temperature over 1 hour. After stirring for a minimum of 3hours, the solids were collected by filtration, washed with toluene andthen dried under vacuum at ˜40° C. to give a recovery of ˜92% of Epo Bactivity. The solids assayed 42.9% w/w Epo B with 16% w/w toluene. Themother liquor contained 66% of the input epothilone A and only 5% of theinput epothilone B.

Example 7C

Pooled heart cuts (200 mL), from the normal phase chromatographyprocedure set forth in Example 7, containing 646 mg of Epo B were slowlyadded to 43 mL of toluene while concentrating under vacuum with a jackettemperature of ˜65° C. to ˜43 mL. Toluene (43 mL) was added under vacuumwhile distillation continued with a jacket temperature of ˜65° C. Theslurry was concentrated to ˜43 mL and was then allowed to cool to ˜20°C. over ˜3 hours The crystals were collected, washed with 2×5 mL tolueneand dried under vacuum (29″ Hg) at ˜40° C. for 30 minutes to give 729 mgof isolated crystalline cake (85.3% w/w Epo B). The HPLC purity was99.77 area % (excluding toluene area %). Residual solvents in the cakewere 15.3% w/w toluene and 0.3% w/w EtOAc. The mother liquor and washcontained only 0.5% of the epothilone B input activity.

An observed PXRD pattern for crystal solvate obtained following themethods described in this step is set forth in FIG. 15 (top pattern),along with a simulated PXRD pattern for a toluene solvate at roomtemperature (bottom pattern). The thermal analysis for this crystalsolvate is set forth in FIG. 16.

Example 8 Preparation of Specific Crystal Forms Example 8A Preparationof epoB-TOβ Preparation of Epothilone B Toluene Solvate

Epo B was dissolved in ˜13 mL of ethyl acetate at ˜40° C. One volume oftoluene was added followed by concentration at a bath temp of <40° C. to9 mL. This was reheated to ˜55° C., followed by the addition of anothervolume of toluene. This was then concentrated to ˜10 mL and allowed tocool to 18° C. The slurry was used for x-ray structure determination.

The molecular structure of the monoclinic unit cell form of epoB-TOβ andPXRD patterns of epoB-Toβ, obtained following the above-describedmethod, are shown in FIGS. 4 and 6, respectively.

Example 8B Preparation of epoB-ANβ Preparation of Epothilone BAcetonitrile Solvate

A solution of essentially pure epothilone B in aqueous acetonitrile(from pooled column fractions resulting from the reverse phasechromatography in example 5) was allowed to evaporate slowly at roomtemperature to yield a crystal slurry from acetonitrile-water. Thecrystal slurry was examined directly by x-ray diffraction.

The molecular structure of the monoclinic unit cell form of epoB-ANβ andthe PXRD patterns of epoB-ANβ, obtained following the above-describedmethod, are shown in FIGS. 2 and 7, respectively.

Example 8C Preparation of epoB-EAβ Preparation of Epothilone B EtOAcSolvate

A solution of epothilone B in 1:1 EtOAc/heptane is concentrated to atarget concentration of ˜190–195 g/L. To this thick slurry of epothiloneB is added with stirring 10 volumes of EtOAc at ˜40° C. The resultingslurry is stirred at 40° C. for 2 hours, is cooled to 0° C. over 5 hoursand is further stirred at 0° C. for a minimum of 5 hours. The slurry isthen filtered and the cake is washed with cold 1:1 EtOAc/heptane. Thecake is dried in the vacuum oven for 5–6 hours to afford the finalepothilone B cake with ˜5–14% EtOAc.

The molecular structure of the monoclinic unit cell form of epoB-EAβ andthe PXRD patterns of epoB-EAβ, obtained following the above-describedmethod, are shown in FIGS. 1 and 5, respectively.

Example 8D Preparation of epoB-IPβ Preparation of Epothilone B IPASolvate

Epothilone B (70 mg) was dissolved in 4 mL of IPA by heating thesolution until a clear solution was formed. This solution was cooled toambient temperature. Any solids formed immediately were removed byfiltration. The clear filtrate was placed in a small vial and coveredwith aluminum foil with a several pinholes. The solvent was allowed toevaporate at ambient temperature very slowly over a period of severaldays until substantial crystal growth was observed. Crystals weresubmitted for X-ray analysis as a wet slurry.

The molecular structure of the monoclinic unit cell form of epoB-IPβ andthe PXRD patterns of epoB-IPβ, obtained following the above-describedmethod, are shown in FIGS. 3 and 8, respectively.

Example 9 Forming Derivative 2 (a Lactam) from Epothilone B (a Lactone)

A tetrabutylammonium azide (TBA azide) solution is prepared by mixingtetrabutylammonium chloride and sodium azide in THF/DMF. The resultingTBA azide solution is recovered by removal of NaCl crystals byfiltration. Catalytic amount of an agent such astris(dibenzyledeneacetone)-dipalladium or the chlorofom adduct of thiscatalyst selected to stabilize an allylic cation, ammonium chloride,epothilone B, and the THF/DMF solution of TBA azide are charged into aflask with agitation. The slurry is deoxygenated by bubbling nitrogenfor about 25 minutes at 0–5° C. Trimethylphospine is added at 0–5° C.The reaction mixture is heated to 32–38° C. and agitation is continuedfor 4–16 hours to produce an amino acid intermediate resulting from thebreakage of the ester functionality. The reaction mixture is cooled to18–24° C. and filtered to remove solids. The solids are washed with THFand the filtrate is combined with the rich filtrate. This solution isadded dropwise over 9–10 hours to THF-DMF slurry of1-hydroxybenzotriazole hydrate,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, andpotassium carbonate at 30–37° C. The resulting mixture is cooled to0–12° C., quenched with water while keeping the temperature <10° C. Themixture is extracted with ethyl acetate three to four times, and thecombined ethyl acetate layers are diluted with cyclohexane (3:1 ethylacetate-cyclohexane ratio) and back extracted with water. The organiclayer is further diluted to 2:1 ethyl acetate-cyclohexane ratio withadditional cyclohexane and passed through an activated charcoalimpregnated cartridge such as Zeta Pad R51SP or R53SP to reduce theamount of residual Pd. Triethylamine (1%) is added to the organicfiltrate and the solution is purified by a short silica-gel filtrationwith 2:1 ethyl acetate-cyclohexane containing 1% triethylamine. Richeluent is collected and concentrated at <37° C. to a final concentrationof 11–14 mL/g. Additional cyclohexane is added and the slurry is heatedat 67–78° C. for 45–60 minutes. The slurry is cooled slowly to about 21°C., filtered and the crystalline solid is washed with 1:1 ethylacetate-cyclohexane. The wet cake is dried in vacuo at <45° C. to yieldcrystalline lactam analogue of epothilone B in about 56M % yield.

Example 10 Forming an Amino-Substituted Epothilone Derivative(Derivative 1) from Epothilone B

Epothilone B is converted to epothilone F by enzymatic hydroxylation ofthe 2-methyl group on the thiazole ring of epothilone B. The conversionis achieved by the action of an actinomycete strain on epothilone B.Actinomycete strains for use in this step are disclosed in U.S. patentapplication Ser. No. 10/321,188, filed Dec. 17, 2002, and WO 00/39276,both of which are incorporated herein by reference.

Epothilone B in ethanol (5% v/w) is added to the microbe, grown in asuitable medium at 16–18° C., and the pH is maintained between 6.9 and7.1 with either 50% w/v sodium hydroxide or 30% w/v sulfuric acid.Bioconversion is continued until the concentration of Epothilone B isreduced to 3–5% of its initial value. A resin such as XAD-16 or SP207capable of adsorbing epothilone F is added to the fermentation tank (5%v/w) and stirred for 16–72 hours at 10–18° C. The fermentation broth isdecanted and the resin is washed with water (2:1 water-resin ratio). Thewash is repeated two more times. Most of the residual water is removedby filtration on a Buchner funnel.

XAD-16 resin, with pre-adsorbed epothilone F, is slurried with water andloaded onto a column. The resin columns are extracted with ethyl acetateand the rich eluate is collected. The aqueous layer is drawn off and therich ethyl acetate fraction is then washed with a 5% sodium bicarbonatesolution and water to remove color. The rich organic fraction isconcentrated under reduced pressure, then passed through a filterprecoated with silica, followed by a 10 μM polish filtration. Theproduct is then distilled under vacuum and primary epothilone F iscrystallized by adding toluene with stirring as an anti-solvent. Therich toluene mixture is further concentrated to reduce the ethyl acetatecontent and more toluene is added. The crystalline slurry is filteredand washed with toluene.

Epothilone F is dissolved in a methylene chloride or methylenechloride/ethyl acetate mixture, then loaded onto a chromatographiccolumn packed with HPLC-grade silica that has been equilibriated with a60–80:40–20 ethyl acetate:n-heptane mixture (v/v).

The product is eluted from the column with either an isocratic or stepgradient of 60–80:40–20 ethyl acetate:n-heptane mixture (v/v), followedby 60–80:40–20 ethyl acetate:n-heptane mixture (v/v). The sample andprocess is monitored via UV detection at 290 nm. The epothilone Fproduct peak is fractionated to minimize closely eluting impurities.Rich pooled fractions are distilled under vacuum to a targetconcentration of approximately 100 g/L. To the slurry of epothilone F,an equal volume of n-heptane is added with stirring. The batch is vacuumredistilled to a target concentration of approximately 100 g/L, ethylacetate is added, and the slurry is maintained at 40° C. The batch iscooled to 2 to −10° C. and maintained for at least 5 hours at thattemperature to crystallize the product from the solution. The resultantslurry is filtered and washed with cool 1:1 ethyl acetate/n-heptanesolution. The final epothilone F cake is dried under vacuum at 35–40° C.

1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 1.8 eq) is added slowly to asuspension of epothilone F and diphenylphosphoryl azide (1.5 eq) intetrahydrofuran (previously dried over 3A MS) and the reaction isstirred at 15–25° C. for 12–24 hours. Trimethylphosphine/tetrahydrofuransolution (1.0 M, 1.1 eq) is slowly added to the reaction mixture. Waterand ammonium hydroxide are added and the mixture is stirred for anadditional 30 minutes. The reaction mixture is diluted with water andthe aqueous phase is extracted with three portions of dichloromethane.The organic phase is then washed with diluted ammonium hydroxide andhalf-saturated sodium chloride solutions, and evaporated to dryness toafford the crude amino derivative (Derivative 1) functionalized on thethiazole methyl group.

The crude product is purified by column chromatography using silica gelpre-treated with 2.5% methanol-0.2% triethylamine-dichloromethane. Thefractions of suitable quality are combined, microfiltered and evaporatedto dryness to afford chromatographed Derivative 1. This material isadded to ethyl acetate and the resulting suspension is heated at 72–75°C. to obtain a solution. Antisolvent n-heptane is added slowly and themixture is allowed to cool slowly in the presence of seeds with stirringat 15–25° C. After cooling and holding at ˜5° C., the resulting solid isisolated by filtration followed by vacuum drying to afford the purifiedcrystalline amino derivative (Derivative 1) in about 70 M % averageyield from Epothilone F.

Example 11 Preparation of Epothilone D (Derivative 3) from Epothilone B

[4S-[4R*,7S*,8R*,9R*,15R(E)]]-4,8-Dihydroxy-5,5,7,9,13-pentamethyl-16-[1-methyl-2-(2-methyl-4-thiazolyl)ethenyl]-1-oxa-13(Z)-cyclohexadecene-2,6-dione[Epothilone D, Derivative 3].

To anhydrous THF (5 ml) at −78° C. under argon was added WCl₆ (198 mg,0.5 mmol) followed by nBuLi (0.625 ml of 1.6 M solution in hexanes, 1.0mmol). The reaction was allowed to warm to room temperature over a 20minute period. An aliquot (0.50 ml, 0.05 mmol) of the tungsten reagentwas removed and added to epothilone B (9.0 mg, 0.018 mmol) under argonand the reaction mixture was stirred for 15 minutes, and then quenchedby the addition of saturated NaHCO₃ (1 ml). The reaction mixture wasextracted with EtOAc (3×1 ml), the combined extracts dried (Na₂SO₄),filtered, and the volatiles were removed under vacuum. The residue waschromatographed with 35% EtOAc/hexanes to give the title compound (7.0mg, 0.014 mmol). MS m/z 492.3 (M⁺+H).

1. A process for isolation of epothilone B from an epothilone-producingmicroorganism comprising: (a) fermenting a strain ofepothilone-producing microorganism in the presence of a resin thatadsorbs epothilone B by hydrophobic interaction; (b) collecting theresin in a water-based medium; (c) extracting the resin with a solventselected to extract epothilone B and to separate it from the water-basedmedium; and (d) crystallizing epothilone B from the extraction phase;wherein said fermentation step further comprises feeding an additivecapable of improving the amount of epothilone B produced as comparedwith the amount of epothilone A produced.
 2. The process of claim 1wherein the crystallized epothilone B from step (d) is substantiallypure.
 3. The process of claim 1 wherein the resin is extracted with apolar solvent.
 4. The process of claim 1 wherein said fermentation stepfurther comprises fermenting said epothilone-producing microorganism inthe presence of skim milk, soy flour, yeast extract, maltrin starch,and/or glycerol.
 5. The process of claim 1 wherein said fermentationstep comprises continuously feeding said additive capable of improvingthe ratio of epothilone B to epothilone A.
 6. The process of claim 1wherein said additive is a propionic acid salt or ester.
 7. The processof claim 6 wherein said additive is sodium propionate, propionic acidmethyl ester or propionic acid ethyl ester.
 8. The process of claim 1wherein the crystallization is conducted to reduce the amount ofepothilone A to about 55% or less of the amount of epothilone A presentafter extraction step (c).
 9. The process of claim 8 further comprising(e) at least a second crystallization step effective to reduce theamount of epothilone A to about 55% or less of the amount of epothiloneA present after crystallization step (d).
 10. The process of claim 1wherein the epothilone-producing microorganism is Sorangium cellulosum.11. The process of claim 10 wherein said microorganism is Sorangiumcellulosum strain ATCC No. PTA
 3880. 12. The process of claim 10 whereinsaid microorganism is Sorangium cellulosum strain ATCC No. PTA
 3881. 13.The process of claim 1 wherein the resin is astyrene/divinylbenzene-based polymer.
 14. The process of claim 13wherein the resin is present in a range of from about 0.2 w/v % to about5.0 w/v %.
 15. The process of claim 1 wherein said step (d) comprises:(i) adding a second solvent in which epothilone B is either not solubleor sparingly soluble; (ii) removing at least a portion of the extractionsolvent; and (iii) transitioning the resultant solvent or solventmixture to a temperature at which epothilone B crystallizes.
 16. Theprocess of claim 15 wherein the extraction solvent is ethyl acetate orMTBE, and the second solvent is toluene.
 17. The process of claim 1further comprising: (f) prior to step (c), washing the resin withaqueous acetonitrile, or aqueous methanol, or an aqueous mediumcomprising a detergent and an amine reagent added in base form, theaqueous medium selected to not elute epothilone B.
 18. The process ofclaim 1, wherein step (c) further comprises polish filtering theepothilone B containing solvent.
 19. The process of claim 1, whereinepothilone B and epothilone A are produced in an epothilone B/A ratio ofat least one.
 20. The process of claim 1, wherein epothilone B andepothilone A are produced in an epothilone B/A ratio of at least 1.5.21. The process of claim 1, wherein epothilone B and epothilone A areproduced in an epothilone B/A ratio in the range of 1.5 to 4.0 .